Alzheimer's disease (AD) is a neurodegenerative disease characterised by the presence of senile plaques and neurofibrillary tangles in the brain. The degree of dementia at death correlates better with neurofibrillary tangle numbers and with neuronal and synaptic loss than with senile plaque counts. The presence of neurofibrillary tangles in neurons results in the death of those neurons, implying that prevention of tangle formation is an important therapeutic goal. The principal protein that forms the neurofibrillary tangle is the microtubule-associated protein, tau, which assembles into filaments that have the appearance of twisting about each other in pairs and are referred to as paired helical filaments (PHF). PHF are present in different locations in degenerating neurons in the Alzheimer brain and when many aggregate in the neuronal cell body, they produce the neurofibrillary tangle (Lee et al., 2001).
Senile plaques have an extracellular central deposit of amyloid β-peptide (Aβ), which is surrounded by dystrophic neurites to form the senile or neuritic plaque. In vitro and in vivo Aβ has been shown to be neurotoxic. Aβ is derived by proteolytic processing of the larger amyloid precursor protein (APP). Much attention has been focused on Aβ production as a therapeutic target because its production is believed to be an early event in AD pathogenesis. This is because mutations in the APP gene, which give rise to autosomal dominant AD, result in either increased overall production of Aβ or in a relative increase in the slightly longer Aβ42 over Aβ40, the former being more amyloidogenic; Aβ42 has two additional hydrophobic amino acids at the C-terminus of 40-residue Aβ40 thereby endowing the peptide with an increased tendency to aggregate and form amyloid fibres. Mutations in two other genes that also cause autosomal dominant AD, presenilin-1 and presenilin-2 (PS1 & PS2) also result in an increase in the ratio of Aβ42 to Aβ40. The belief that Aβ deposition in the brain precedes the appearance of neurofibrillary tangles has been the basis of the amyloid cascade hypothesis but it has been uncertain whether tangles are important in pathogenesis or are only an unimportant epiphenomenon. This has been changed by the discovery of mutations in the gene for tau in some other related neurodegenerative diseases.
The mechanism by which Aβ kills neurons in the brain has still to be established. Many studies of Aβ toxicity have been conducted in tissue culture using rat brain neuronal cultures. We have shown that exposure of both foetal rat and human brain neuronal cultures to aggregated Aβ induces within 2 to 10 minutes increases in the phosphotyrosine content of several proteins including tau (Williamson et al., 2002). We have also shown that this treatment results in activation of the tyrosine kinases FAK and Fyn, the latter being a member of the src family of tyrosine kinases. This tyrosine phosphorylation of tau was prevented by inhibitors that act on the src family of tyrosine kinases and act on c-Abl.
It has previously been reported that increased levels of Fyn are associated with neurons containing abnormally phosphorylated tau in AD brain (Shirazi and Wood, 1993) and we have demonstrated using antibodies that recognise phosphotyrosine that PHF-tau from AD brain contains phosphotyrosine (Williamson et al., 2002). There are five potential sites for tyrosine phosphorylation in tau, these are residues 18, 29, 197, 310 and 394, based upon the numbering of residues in the longest human brain isoforms of tau of 441 amino acids. We have shown in vitro that Fyn and Lck, both src family kinases, phosphorylate recombinant human tau and phosphotyrosines 18, 197, 310 and 394 were positively identified in one or more of their respective tryptic peptides, from sequence information of fragmented peptides (Scales et al., 2002).
Neurons in brain slices from transgenic mice in which the Fyn gene has been disrupted are resistant to Aβ toxicity (Lambert et al., 1998). Thus, there is evidence that activation of Fyn may be involved in Aβ toxicity.
It has been reported that Aβ treatment of microglia in culture results in activation of several other tyrosine kinases, namely Syk, Lyn and FAK (McDonald et al., 1997) and, as mentioned above, we have found that FAK is also activated in primary neurons exposed to Aβ (Williamson et al., 2002). Syk has been reported to phosphorylate α-synuclein on tyrosine, α-synuclein being the principal protein of Lewy bodies which are the pathological hallmark of Parkinson's disease and are also present in up to 70% of AD brains (Negro et al., 2002). Finally, we have found that the protein tyrosine kinase Abl phosphorylates tau in co-transfected cells and Abl is implicated in activation of the serine/threonine protein kinase cdk5, which is regarded as a pathogenically important tau kinase that phosphorylates many residues in tau that can alternatively be phosphorylated by GSK-3 (Zukerberg et al., 2000). Thus, there is the strong possibility that tau is a substrate for various tyrosine kinases and that these need to be considered in the context of the possible pathogenesis of the tauopathies.
The presence of intraneuronal deposits of tau in the form of typical neurofibrillary tangles in AD or other morphologically distinct tau aggregates in a number of other neurodegenerative diseases, is the basis for grouping these conditions as tauopathies. Thus, in addition to AD, the main examples of the tauopathies are frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, corticobasal degeneration, and multisystem atrophy (MSA). The intracellular tau deposits (usually neuronal but sometimes also glial) are filamentous and in a hyperphosphorylated state compared to the phosphorylation of tau in control human brain. In the case of AD, this hyperphosphorylated tau is often referred to as PHF-tau because it is derived from the PHF.
Other than for AD, deposits of Aβ in the brain are either absent or minimal in these other tauopathies. There are some tauopathy pedigrees with autosomal dominant disease in which the causative gene has been identified as the tau gene and although cases with the same mutation may present with apparently different diseases, they invariably have tau deposits in the brain and are mostly of the FTDP-17 variety. Thus, the finding of mutations in the tau gene which result in disease and deposition of tau aggregates in neurons is compelling evidence for the primary pathogenic importance of tau deposition in all of these conditions, including AD, whatever the primary cause of disease. Therefore, the amyloid cascade hypothesis is borne out by the discovery of tau mutations and confirms that indeed neurofibrillary tangle formation may well be subservient to Aβ deposition in AD, but that in the other tauopathies lacking Aβ deposits, then some other primary event must trigger the tau pathology. Tau abnormalities and deposition are therefore important therapeutic targets for all tauopathies, including AD.
Tau is a phosphoprotein, the function of its phosphorylation remaining to be unequivocally established. However, increased phosphorylation of tau on multiple serine and threonine residues reduces the ability of tau to promote microtubule assembly and to stabilise assembled microtubules, effects that have been demonstrated both in vitro and in cells. Many studies have shown that PHF-tau from AD brain is more heavily phosphorylated on serine and threonine than tau from control brain. This has been demonstrated partly by protein sequencing and partly by demonstrating that certain monoclonal antibodies only label either PHF-tau or alternatively they label non-phosphorylated tau and not PHF-tau; the epitopes for many of these antibodies have been mapped to particular phosphorylated residues present in PHF-tau and absent from, or present at lower levels in, control brain tau. The pathological tau from most other cases of other tauopathies seems to be similarly hyperphosphorylated to PHF-tau.
These findings strongly imply that similar abnormalities in regulating phosphorylation of tau are shared by all the tauopathies including AD. Since phosphorylation of proteins is effected by protein kinases and dephosphorylation by protein phosphatases, identifying the protein kinases and phosphatases for tau is important because these enzymes are potential therapeutic targets for these diseases.
As mentioned above, there are five tyrosines in human brain tau. It has been reported that Fyn phosphorylates tau in non-neuronal co-transfected cells and that tyrosine 18 is the preferred phosphorylation site (Lee et al., 1998). We have reported that PHF-tau isolated from Alzheimer brain is phosphorylated on tyrosines and others have identified tyrosine 18 as one site of phosphorylation (Williamson et al., 2002; Lee et al., 2004).
Cultured neurons from transgenic mice in which the tau gene has been disrupted, such that these animals no longer express the tau protein, are resistant to exposure to Aβ and do not die (Rapoport et al., 2002). This requirement of tau for Aβ to be neurotoxic has been confirmed in experiments in which neurons treated with antisense oligonucleotides to reduce expression of tau were resistant to the neurotoxic effects of Aβ exposure (Liu et al., 2004).
It remains a considerable problem in the art in identifying the enzymes responsible for causing phosphorylation of paired helical filament tau and the sites phosphorylated by those enzymes.